Cell surface receptors are an important class of proteins involved in cellular functioning because they are the primary mediators of cell to cell communication. In particular, G protein-coupled receptors (GPCRs) are an important category of cell surface receptors. The medical importance of these receptors is evidenced by the fact that more than 60% of all commercially available prescription drugs work by interacting with known GPCRs.
In their resting state, the G proteins, which consist of alpha (α), beta (β) and gamma (γ) subunits, are complexed with the nucleotide guanosine diphosphate (GDP) and are in contact with the receptors to which they are coupled. When a hormone or other first messenger binds to receptor, the receptor changes conformation and this alters its interaction with the G protein. This spurs the α subunit to release GDP, and the more abundant nucleotide guanosine triphosphate (GTP) replaces it, activating the G protein. The G protein then dissociates to separate the α subunit from the still complexed beta and gamma subunits. Either the Gα subunit, or the Gβγ complex, depending on the pathway, interacts with an effector. The effector (which is often an enzyme) in turn converts an inactive precursor molecule into an active “second messenger,” which may diffuse through the cytoplasm, triggering a metabolic cascade. After a few seconds, the Gα converts the GTP to GDP, thereby inactivating itself. The inactivated Gα may then reassociate with the Gβγ complex.
Hundreds, if not thousands, of receptors convey messages through heterotrimeric G proteins, of which at least 17 distinct Gα subunit forms have been isolated. Most G protein-coupled receptors are comprised of a single protein chain that is threaded through the plasma membrane seven times. Such receptors are often referred to as seven-transmembrane domain receptors (STRs). More than a hundred different GPCRs have been found, including many distinct receptors that bind the same ligand, and there are likely many more GPCRs awaiting discovery.
The mating factor receptors of yeast cells (STE2 and STE3) span the membrane of the yeast cell seven times and are coupled to yeast G proteins. Heterologous GPCRs can be expressed in yeast cells and can be made to couple to yeast G proteins resulting in the transduction of signals via the endogenous yeast pheromone system signaling pathway which is normally activated by STE2 or STE3. In some cases, such heterologous receptors can be made to couple more effectively to the yeast pheromone system signaling pathway by coexpressing a heterologous G protein α subunit (e.g. U.S. Pat. No. 5,482,835 to King et al), by expressing a chimeric G protein subunit (e.g. WO 94/23025), or by expressing a chimeric G protein-coupled receptor (e.g., U.S. Pat. No. 5,576,210 issued to Sledziewski et al.).
The βγ subunits of the activated G protein stimulate the downstream elements of the pheromone response pathway, including the Ste20p protein kinase, and a set of kinases that are similar to MEK kinase, MEK (MAP kinase kinase), and MAP kinase of mammalian cells and are encoded by the STE11, STE7, and FUS3/KSS1 genes, respectively (Whiteway et al. 1995. Science. 269:1572).
Members of the family of chemotactic cytokines, which have been proposed to be named “chemokines” for short, are being identified as vital initiators and promulgators of inflammatory and immunological reactions (Oppenheim et al. (1991) Annu Rev Immunol 9:617). The chemokines range from 8 to 11 kD in MW, are active over a 1 to 100-ng/ml concentration range, and are produced by a wide variety of cell types. They are induced by exogenous irritants and endogenous mediators such as IL-1, TNF, PDGF, and IFN-γ. The chemokines bind to specific cell surface receptors with a KD of 0.4 to 4 nM. These chemokines can be considered “second-order” cytokines that appear to be less pleiotropic than “first-order” proinflammatory cytokines because they are not potent inducers of other cytokines and exhibit more specialized functions in inflammation and repair. As shown in Table 1, some of the chemokines have been assigned to a “chemokine α” subset based on their gene cluster on chromosome 4 (q12–21) and based on the fact that the first two of their four cysteine groups are separated by one amino acid (C—X—C).
TABLE 1Properties of the chemokine α subfamilyChemotactic orExogenousEndogenoushaptotacticCytokineCell sourcesstimulantsinducersresponsesMajor activitiesIL-9MonocytesEndotoxinIL-1NeutrophilsActivates PMNNeutrophilsMitogensTNFBasophils↑ NeutrophilFibroblastsParticulatesIFN-γUnstimulatedadhesionEndothelialVirusescostimulantT cells↓ BasophilcellsIL-3MelanomahistamineKeratinocytesCells↑ KeratinocyteLarge granulargrowthlymphsAcuteT lymphocytesInflammationGRO-αβγ/MonocytesEndotoxinIL-1NeutrophilsDegranulatesmu/KCFibroblastsTNFPMNmuMIP-2αβEndothelial↑ Melanomacellscell growth↑ FibroblastgrowthAcuteinflammationCTAP III/MonocytesPlateletFibroblasts↑ FibroblastβTGPlateletsactivatorsgrowthβTG/NAP-2MonocytesPlateletNeutrophilsActivates PMNPlateletsactivatorsPF-4PlateletsPlateletFibroblasts↑ FibroblastactivatorsgrowthReversesimmunesuppression↑ I-CAM1 onE.C.IP-MonocytesEndotoxinIFN-γMonocytes↑ Chronic10/muCCRG-2FibroblastsActivated TinflammationEndotheliallymphocytescellsKeratinocytesENA-78Epithelial cellsIL-1NeutrophilsActivates PMNTNFThis chemokine α group includes IL-8, melanoma growth-stimulating activity (MGSA/GRO), platelet factor 4 (PF-4), β thromboglobulin (βTG), IP-10, and ENA-78.
IL-8 is produced by many cell types including NK cells and T lymphocytes in response to exogenous stimuli such as polyclonal mitogens, injurious stimuli, and infectious agents, as well as proinflammatory cytokines such IL-1 and TNF. IL-8 is a chemoattractant of neutrophils, basophils, and a small proportion (10% or less) of resting CD4+ and CD8+ lymphocytes. IL-8 additionally activates neutrophil enzyme release. IL-8 is also haptotactic for melanocytes and is a comitogenic stimulant of keratinocytes.
IL-8 promotes the adherence of neutrophils to endothelial cells. IL-8 does so by inducing neutrophils to express β2 integrins. Neutrophils then extravasate by moving between the endothelial cell junctions and through the basement membrane to accumulate in the tissues. Intracutaneous injections of IL-8 cause a rapid local neutrophilic infiltration peaking with 3 hours. Intravenous administration of IL-8 does not induce systemic sequelae of elevation of acute-phase proteins or fever but does induce a neutrophilia. Intravenous administration of IL-8 also specifically reduces local peripheral inflammatory responses to IL-8, ƒMLP, and C5a (Hechman et al. (1990) FASEB J 4:890). This transient anti-inflammatory effect of IL-8 probably can be attributed to desensitization of neutrophils by systemically distributed IL-8.
Two distinct but homologous (70% at the amino acid level) receptors for IL-8 have been cloned. The IL-8 receptors are members of the rhodopsin receptor family and have a seven transmembrane spanning region (Holmes et al. (1991) Science 253:1278; Murphy et al. (1991) Science 253:1280). The receptors are probably coupled to G-proteins, transduce phosphoinositol hydrolysis, and are capable of rapid elevation of diacylglycerol and cytosolic Ca2+ levels, which may lead to activation of protein kinase C (Thelen et al. (1988) FASEB J 2:2702). IL-8 receptors are expressed by neutrophils, which display both types of IL-8R and their expression is unregulated by G-CSF (A. Lloyd et al., unpublished results). Mature neutrophils express about 20,000 receptors per cell. Myelocytic lines and basophils express several thousand receptors per cell.
IL-8 like molecules have been identified in rabbits, sheep, and other species. IL-8 is in the circulation of patients with systemic inflammatory reactions or severe trauma. IL-9 has readily been detected in inflammatory sites such as in the synovial fluid in rheumatoid arthritis (Brennan et al. (1990) Eur J. Immunol 20:2141), extracts of psoriatic skin (Schroder et al. (1986) J Invest Dermatol 87:53, and in the circulation of patients in septic shock (Van Zee et al. (1991) J Immunol 146:3478–3482). Thus IL-8 is implicated as a major participant in acute as well as more prolonged inflammatory reactions.
MGSA, as its name implies, was first discovered as a factor that accelerated the growth of melanoma cell lines and also as a product of oncogene transfected cell lines (GRO). MGSA/GRO competes for the type II, but not type I, IL-8 receptor on myelocytic cells (Lee (1992) J Biol Chem 267:16283–16287) and is also a potent chemoattractant, as well as activator of neutrophils. MGSA as well as IL-8 has been extracted from psoriatic tissues (Sticherling et al. (1991) J Invest Dermatol 96:26).
GRO has three variants (α, β, and γ), which exhibit about 95% homology in their amino acid sequences. They are probably homologues of murine macrophage derived KC, macrophage derived inflammatory peptides MIP-2α and MIP-2β. Murine MIP-2α and MIP-2β both compete with equal affinity for type II receptors for IL-8 and chemoattract human as well as murine neutrophils (Lee et al. (1992) J. Biol Chem 267:16283–16287). MIP-2 is also reported to degranulate murine neutrophils, resulting in the release of lysosomal enzymes. Local in vivo injections of MIP-2 results in neutrophil accumulation and MIP-2 has been isolated from sites of wound healing. MIL-2 is a costimulator of hematopoietic colony formation by CSF-1 and GM-CSF, but the in vivo relevance of this observation remains to be established. It is most likely that GRO/MIP-2 inflammatory activities overlap considerably with those of IL-8, and GRO is therefore probably also a major inflammatory mediator.
PF-4 and CTAP III, the precursor of βTG, are both present in platelet granules and are released by inducers of platelet aggregation. Consequently, they become available at sites of injury, hemorrhage, and thromboses. Both are reported to chemoattract and to stimulate fibroblasts, presumably for repair purposes. In addition, a 70 amino acid breakdown product of βTG known as neutrophil attracted peptide 2 (NAP-2) is a chemoattractant and activator of neutrophils, albeit at 100-fold higher concentrations that IL-8. NAP-2 also competes for the type II IL-8 receptor with about one-hundredth of the affinity of IL-8 (Leonard et al. (1991) J. Leukoc Biol 49:258). However, since at the site of platelet aggregation, high levels of NAP-2 can be released, it is thought to be an active participant in attracting inflammatory cells to such sites.
ENA-78 is the most recently cloned member of the chemokine α subfamily (Walz et al. (1991) J Exp Med 174:1355). ENA-78 is produced by an epithelial cell line in response to IL-1 and TNF. In cross-desensitization experiments, ENA-78 also utilizes the type II receptor for IL-8 and GRO and is a chemoattractant and activator of neutrophils.
IP-10 is produced by macrophages, endothelial cells, and keratinocytes in response to IFN-γ. The pathophysiological functions of IP-10 remain unclear, but antibodies to IP-10 react with many cell types present at sites of delayed hypersensitivity reactions and IP-10 has been extracted from psoriatic plaques (Gotlieb et al. (1988) J Exp Med 168:941). Thus IP-10 can presumably be produced by many cell types and probably participates in chronic inflammation and delayed hypersensitivity responses.
A stable recombinant human IP-10 was recently produced by Dr. K. Matsushima (personal communication). We have shown that this rhIP-10 is a moderately potent in vitro chemoattractant of human monocytes, but not neutrophils. In addition, this IP-10 also is a moderately potent chemoattractant for previously activated CD4 and CD8 T lymphocytes and promotes adhesion of lymphocytes to endothelial cells (D. Taub et al., unpublished results). These observations predict that IP-10 will probably be a participant in chronic cell-mediated inflammatory reactions.
There are two known human subtypes of the IL-8 receptor. Subtype A (CXCR1) binds IL-8 with high affinity (Kd=0.1 nM) but shows very low affinity binding to GROα and NAP-2 (Kd>100 nM). Subtype B (CXCR2) binds all three ligands with high affinity.
Overall, the chemokine family members appear to be very potent and pivotal chemoattractants and activators of inflammatory cells and fibroblasts. The aforementioned inflammatory cytokines have myriad effects on cell growth and differentiation. Some of these effects are indirect and based on the capacity of cytokines to induce the production of a cascade of other cytokines. Therapeutic usefulness of cytokines and their inhibitors is growing and should accelerate.
In recent years drug discovery has been advanced by expression of heterologous receptors in living cells. However, due to the complexities inherent in such heterologous expression studies, the development of reliable assays to search for modulators of these receptors has presented particular challenges. For example, it is often difficult to obtain sufficient expression of heterologous G protein-coupled receptors or to achieve functional integration of the G protein-coupled receptor into a signaling pathway. Price et al. have reported the functional coupling of a rat A2a adenosine receptor into the yeast pheromone response pathway (Price et al. (1996) Molec. Pharmacol. 50:829–837), but only achieved effective coupling when they coexpressed the native yeast G protein GPA1, expressed from a plasmid, in the yeast cells. They were unable to achieve effective coupling using either a mammalian Gα subunit protein or a chimeric Gα subunit protein expressed in the yeast cells. The development of expression and coupling systems for expression of modified G protein coupled receptors which display altered ligand binding and/or coupling in host cells would be of tremendous benefit in the development of improved drug screening assays for modulators of G protein coupled receptors.